Fig. 6: Evolution of the CYP76AK subfamily.

a Syntenic relationships among CYP76AK genes in Salvia genomes revealed the divergence of CYP76AK clades. The phylogenetic tree in the left panel represents the phylogeny of six Salvia species with N. cataria as the outgroup. WGD events are indicated by red dots. Microsynteny of CYP76AK genes in the synteny blocks. Red polygons show syntenic relationships between CYP76AK genes, and gray polygons show other genes. Anc-CYP76AK, Ancestral CYP76AK gene. b Reconstruction of ancestral CYP76AK proteins. Ancestral nodes (ancNodes) 1−6 show the resurrected enzyme of each major branch point of the CYP76AK phylogenetic tree. Functional characters of each branch are distinguished by different colors. c, d Ancestral function of CYP76AKs. Extracted ion chromatograms (EICs) showing in vitro catalytic activity of each ancestral CYP76AK using 11-hydroxyferruginol and 11-hydroxysugiol as the substrate, respectively. e Evolution of ATDs biosynthesis in Salvia. The phylogenetic tree shows the speciation of the clades of Salvia, and the diamonds represent the predicted ancestral metabolic traits for each speciation node. Square frames represent the realistic metabolic traits of each Salvia clade as shown in Fig. 3. Green, red, and light blue represent the existence of carnosic acid, tanshinones, and 20-keto ATDs, respectively. The time scale on the left reflects the time of speciation for each clade. Branches highlighted in light blue indicate clades in which the ancestral metabolic traits have been retained. f Chronology of CYP76AK genes. The phylogenetic tree represents the evolution of CYP76AK clades according to the MCMCtree model. The speciation time for each clade is labeled. Branches highlighted in blue indicate CYP76AK clades that have retained their ancestral catalytic functions, and branches highlighted in red indicate clades that have undergone a loss of function. The arrows at the bottom indicate the CYP76AK clade that contributes to producing the type of ATDs in the corresponding lineage. Green, red, and indigo gray arrows indicate the biosynthesis of carnosic acid, tanshinones, and 20-keto ATDs, respectively. Source data are provided as a Source Data file.